Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and comprises two homodimeric subunits, R1 and R2. 2'-Azido-2'-deoxyuridine 5'-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism-based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen-centered radical. The X-band EPR spectrum of this stable radical species exhibits a triplet hyperfine interaction of ~25 G arising from one of the three nitrogens of the azide group of N3UDP and a doublet hyperfine interaction of 6.3 G, which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen-centered radical have resolved the peaks corresponding to all three principal g-values: g11 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved ( = 31.0 G) and upper limits (~ 5 G) can be placed on both and . Comparison of these g and A values with those of model systems in the literature suggests a structure for the radical, -X-N-S-CH2-, in which -S-CH2- is part of a cysteine residue of R1 and X is either an unprotonated sulfur, oxygen or carbon. Use of an E. coli strain that is auxotrophic for cysteine and contains the ribonucleotide reductase gene allowed [b-2H]-cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -S-CH2-component of the proposed structure. These results establish that the nitrogen-centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439, 462, 754 & 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen-centered radical is proposed in which X is derived from 3'-keto-2'-deoxyuridine 5'-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3'-hydroxyl oxygen of the deoxyribose.